Characterization of mutations affecting myofibrillogenesis in the zebrafish Brachydanio rerio

As skeletal muscle cells undergo terminal differentiation they elongate and build contractile machinery oriented with the long axis of the cell. How does the cell construct this complex assembly of cytoskeletal components? From previous in vitro evidence, these myofibrils have been hypothesized to assemble on an oriented scaffold of cortical actin filament bundles, or "Stress-Fiber-Like-Structures" (SFLS). To address this problem, I describe the early events of myofibrillogenesis in the developing embryo of the zebrafish, Brachydanio rerio. I have ultrastructurally characterized an identified population of skeletal muscle cells that elongate and undergo myofibrillogenesis at 14 hours after fertilization. These cells contained SFLS. In zebrafish this process can be examined using mutational analysis. I describe two gamma-ray induced lethal mutations, fub-1(b45) and fub-1(b126), that were found in systematic screens for nonmotile embryos and which disrupt myofibrillogenesis. These recessive mutations each segregate as a single locus. The two mutations fail to complement and have similar gene-centromere distances indicating that they may be allelic. They are fully penetrant, and the b45 allele has a more severe phenotype. Data from mosaic analyses suggest that fub-1(b45) is cell-autonomous. Skeletal muscle cells in homozygotes elongate normally, but have severely disoriented myofibrils as revealed by polarized light- and transmission electron microscopy. Ultrastructural studies and specific probes for F-actin reveal that well-ordered SFLS are absent from, or reduced in developing mutant muscle cells. These studies are consistent with the hypothesis that SFLS constitute an orienting scaffold for myofibrils.